In Escherichia coli (E. coli), programmed cell death is mediated through “addiction modules” consisting of two genes, one of which encodes a stable toxic protein (toxin) and the other encodes a short-lived antitoxin. The toxin and the antitoxin are coexpressed from an operon and interact with each other to form a stable complex and their expression is auto-regulated either by the toxin-antitoxin complex or by the antitoxin alone. When their co-expression is inhibited by stress conditions, for example, the antitoxin is degraded by proteases, enabling the toxin to act on its target. Such genetic systems for bacterial programmed cell death have been reported in a number of E. coli extrachromosomal elements for the so-called postsegregational killing effect. When bacteria lose the plasmids or other extrachromosomal elements, the cells are selectively killed because unstable antitoxins are degraded faster than their cognate stable toxins. Thus, the cells are addicted to the short-lived antitoxins since their de novo synthesis is essential for cell survival.
The condensed single protein production (cSPP) system was developed based on the endoribonuclease activity of an Escherichia coli toxin called MazF, which selectively cleaves cellular mRNAs at the ACA codon sequence. Upon induction of MazF, protein synthesis is completely inhibited, and as a result, cell growth is also completely arrested. However, MazF induced cells are in a quasidormant state, as they are metabolically fully active, producing ATP, amino acids, and nucleotides. Most significantly, in the quasidormant cells, machineries for protein synthesis and mRNA production are also fully functional. Therefore the MazF-induced quasidormant cells are still capable of synthesizing a protein of interest without producing any other cellular proteins, if the mRNA for the protein is engineered to have no ACA sequences. This system is thus termed the single protein production (SPP) system.
One of the most remarkable advantages of the SPP system is that the cell culture can be highly condensed without affecting protein yields. Using this cSPP system, one can achieve a cost savings of as much as 97.5% by condensing a culture 40 fold. This is particularly valuable when highly expensive isotopes or isotope labeled compounds, such as amino acids and glucose, are used for the preparation of protein samples for structural study by nuclear magnetic resonance (NMR) spectroscopy. Furthermore, by use of the cSPP system, amino acid analogues or D20, which is toxic in conventional protein production systems, reducing protein yields, is not toxic, hardly affecting the final protein yields. However, one drawback of the current cSPP system is the use of IPTG (isopropyl β-D-thiogalactopyranoside) as an inducer for both MazF and a target protein, such that the target protein is also produced at the same time as MazF. Since isotopes or isotopelabeled compounds are added 2 to 3 hours after the addition of IPTG to avoid their incorporation into cellular proteins, nonisotope labeled target protein is also produced during this preincubation period, resulting in a higher background of unlabeled target protein, which may be as high as 20% of the final yield of the target protein produced. The combination of both tetracycline and IPTG inducible systems was employed to separate the inductions of target protein and MazF, respectively. The main disadvantage of this system is that the expression level of target protein critically depends upon tetracycline concentration. The amounts of tetracycline being added to the cells for induction of target protein significantly affect the level of target protein synthesis, especially in the condensed SPP system. Therefore, a highly precise and accurate optimization of the tetracycline level is required for consistency in the expression of target proteins. Thus there remains a need for a more accurate and consistent method of single protein production.